This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information
IFI30 is a gene most commonly known as GILT (gamma-interferon-inducible lysosomal thiol reductase) and its primary role is to reduce disulfide bonds of endocytosed proteins in acidic environments, which facilitates the proteins' unfolding and degradation (West & Cresswell, 2013). Various studies focused on the role of IFI30 and antigen processing have shown results confirming that the influence of IFI30 on the "peptide repertoire" can change the type of immune response and thus affect "central tolerance" (West & Cresswell, 2013). The enzyme produced by this gene is present in antigen-presenting cells and holds a major influence in MHC class II-restricted antigen processing as shown in Figure 1(West & Cresswell, 2013). Furthermore, thiol reductases that are related to IFI30 have been found in various species that predate the adaptive immune system suggesting that IFI30 has functions that differ from antigen processing (West & Cresswell, 2013). Balce et. al. (2014) also found that in addition to reducing disulfide bonds of endocytosed proteins, IFI30 maintains phagosomal proteolytic activity, specifically in activated macrophages.
Figure 1
Figure 1: Diagram of MHC class II antigen processing. The MIIC is the MHC class II compartment, where proteases degrade endocytosed proteins. IFI30 specifically breaks down disulfide bonds of these proteins. Image courtesy of Nature Reviews: Immunology.
Methods/Approach
The human protein sequence for IFI30 (ENSP00000384886) was attained through the Ensembl database. This protein sequence was used as a query in a BLAST against the whale shark protein database with the Georgia Aquarium Galaxy server. Predicted protein hits were chosen based on their alignment lengths, e-values, and % identities along with query coverage, and their sequences were obtained through the Galaxy server. These predicted protein sequences were then used as queries in subsequent BLASTs against the human protein database through NCBI.
Orthologs were searched for by using the human protein sequence for IFI30 (ENSP00000384886) as the query sequence for BLASTs against the protein databases of other species.
A phylogenetic tree was created with CLUSTALW using the top predicted protein hits of the BLASTs against the protein databases of other species as well as the whale shark using the human protein sequence as the query. The best hits were recorded and analyzed.
Searching for IFI30 in the Whale Shark
The human IFI30 protein sequence was used as the query in a BLAST against the whale shark protein database. The BLAST resulted in only two predicted proteins as shown in Figure 2 and Table 1.
Figure 2
Figure 2: The results from a BLAST using the human IFI30 protein sequence as the query against the whale shark protein database through the Georgia Aquarium Galaxy server.
Table 1
Whale Shark ID
E-value
Alignment Length
Predicted Protein Length
% Identity
g13114.t1
6e-04
22
85
40.91
g37936.t1
9e-05
22
96
45.45
Table 1: The results from a BLAST using the human IFI30 protein sequence as the query against the whale shark protein database. These were the only two predicted proteins that resulted from the search.
The g13114.t1 whale shark sequence was used as the query in a BLAST against the human protein database. The best hit was a coiled-coil domain-containing protein 71 [Homo sapiens] (NP_075054.3) with a query cover of 85%, an e-value of 2e-19, and a percent identity of 52%. The g37936.t1 whale shark sequence was also used as the query in a BLAST against the human protein database, but the best hit did not contain any putative conserved domains. This whale shark sequence was also used in subsequent BLASTs against the elephant shark, zebra fish, and clawed frog protein databases but returned no putative conserved domains and no similar proteins matches for best hits.
The g13114.t1 whale shark sequence was also used as the query in a BLAST against the elephant shark protein database in order to further establish its relationship to the coiled-coil domain. The best hit matched the best hit from the BLAST against the human protein database. The elephant shark predicted protein hit, coiled-coil domain containing protein 71 (XP_007888756.1) had a query cover of 83%, an e-value of 2e-28, and a percent identity of 66%. This elephant shark sequence was then used as the query in a BLAST against the human protein database. This BLAST resulted in the same best hit in the human protein database as the BLAST with the whale shark sequence as the query.
Protein Domains
Since only the g13114.t1 whale shark protein sequence returned hits against the human and elephant shark protein databases with the coiled-coil domain conserved, it seems likely that whale sharks have the coiled-coil domain (as seen in Figure 3).
Figure 3
Figure 3: The putative conserved domain resulting from a BLAST using the g13114.t1 whale shark protein sequence against the human protein database.
Coiled Coil-type proteins are a structural domain of proteins. They usually consist of two or more alpha helices in a helical twist (Coiled coil, 2014). They are involved in many important biological functions, primarily the regulation of gene expression. They are also involved in HIV infection. A spring loaded mechanism constructed of gp41 protein forms a coiled coil, essentially forming a "trimer of hairpins" (or six helix bundle) that facilitates membrane fusion by pulling cell membranes closer to each other. The HIV virus then enters the cell and begins to replicate (Nash 2015).
Innate immune sensors that detect foreign molecules consist of N-terminal coiled coil, or TIR (toll/interleukin-1 receptor) domains along with LRRs (leucine-rich repeats) (Rairdan 2008). Coiled-coil proteins are involved in downstream signaling for the immune system (Nash 2015).
Orthologs
In order to find IFI30 orthologs in other species, the human IFI30 protein sequence was used as the query in BLASTs against the protein sequences of mice, zebra fish, elephant sharks, clawed frogs, yeast, and fruit flies. The best hits for each of the BLASTs are shown in Table 2.
Interferon, gamma-inducible protein 30 precursor [Xenopus (Silurana) trpicalis]
NP_001017196.1
4e-68
77%
50%
256
Yeast
Ecm29p [Saccharomyces cerevisiae S288c]
NP_011833.1
1.3
10%
52%
1868
Fruit fly
CG41378, isoform C [Drosophila melanogaster]
NP_001104369.2
1e-22
70%
30%
196
Table 2: The best hits of BLASTs against numerous species using the human IFI30 protein sequence as the query. These species include mice, zebra fish, elephant sharks, clawed frogs, yeast, and fruit flies. The top predicted protein hits are recorded here.
Phylogeny
A phylogenetic tree (Figure 4) was constructed through ClustalW using the best hits from the different species listed in Table 2 from the BLAST searches with the human IFI30 protein as query sequence.
Figure 4
Figure 4: A phylogenetic tree showing the best hits of the human IFI30 protein in mice, elephant sharks, clawed frogs, zebra fish, fruit flies, whale sharks (g13114.t1), and yeast.
It is interesting that the whale shark sequence g13114.t1 groups in between the fruit fly and yeast. The top hits from the fruit fly and the yeast protein databases were used as query sequences in BLASTs against the human protein database. The top hits yielded coiled-coil domain containing proteins. Therefore, we were confident that whale sharks conserved this protein domain. However, its distance from the human protein sequence as shown in Figure 4 implies that the whale shark predicted protein sequence is not very similar to the human IFI30 protein sequence.
Conclusions
We were not able to identify an ortholog of the IFI30 protein in the whale shark, but we were able to identity a conserved domain: the coiled-coil domain. Since the BLAST against the human protein database using the g13114.t1 whale shark sequence as a query did not return the IFI30 protein sequence as the best hit, we were not confident that the whale shark has this specific protein as an ortholog. However, we were able to identify the conservation of the coiled-coil domain through subsequent BLASTs with the elephant shark protein database, and we were able to establish a relationship. Therefore, whale sharks may not benefit from the activity of gamma-interferon-inducible lysosomal thiol reductase, which facilitates protein degradation. Further research could look into the way the whale shark's immune response is different from humans since it seems to lack the gene that encodes for the lysosomal thiol reductase.
References:
Balce, D. R., Allen, E. R., McKenna, N., & Yates, R. M. (2014). γ-Interferon-inducible Lysosomal Thiol Reductase (GILT) Maintains Phagosomal Proteolysis in Alternatively Activated Macrophages. The Journal Of Biological Chemsitry,289(46), 31891-31904. doi:10.1074/jbc.M114.584391
Rairdan, G. J., Collier, S. M., & Sacco, M. A. (2008). The Coiled-Coil and Nucleotide Binding Domains of the Potato Rx Disease Resistance Protein Function in Pathogen Recognition and Signaling. Plant Cell,20(3), 739-751. Retrieved April 13, 2015.
Maekawa, T., Cheng, W., & Spiridon, L. N. (2011). Coiled-coil domain-dependent homodimerization of intracellular barley immune receptors defines a minimal functional module for triggering cell death. Cell Host Microbe,9(3), 187-199. Retrieved April 13, 2015.
Neefjes, J., Jongsma, M., Paul, P., & Bakke, O. (2011, December). The basic MHC class II antigen presentation pathway [Jacques Neefjes, Marlieke L. M. Jongsma, Petra Paul & Oddmund Bakke]. Retrieved April 14, 2015, from http://www.nature.com/nri/journal/v11/n12/fig_tab/nri3084_F3.html
[Image]
West, L. C., & Cresswell, P. (2013). Expanding roles for GILT in immunity. Current Opinion in Immunology,25(1), 103-108. doi:10.1016/j.coi.2012.11.006
IFI30
(Interferon, gamma-inducible protein 30)This Project
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.Background Information
IFI30 is a gene most commonly known as GILT (gamma-interferon-inducible lysosomal thiol reductase) and its primary role is to reduce disulfide bonds of endocytosed proteins in acidic environments, which facilitates the proteins' unfolding and degradation (West & Cresswell, 2013). Various studies focused on the role of IFI30 and antigen processing have shown results confirming that the influence of IFI30 on the "peptide repertoire" can change the type of immune response and thus affect "central tolerance" (West & Cresswell, 2013). The enzyme produced by this gene is present in antigen-presenting cells and holds a major influence in MHC class II-restricted antigen processing as shown in Figure 1(West & Cresswell, 2013). Furthermore, thiol reductases that are related to IFI30 have been found in various species that predate the adaptive immune system suggesting that IFI30 has functions that differ from antigen processing (West & Cresswell, 2013). Balce et. al. (2014) also found that in addition to reducing disulfide bonds of endocytosed proteins, IFI30 maintains phagosomal proteolytic activity, specifically in activated macrophages.Figure 1
Figure 1: Diagram of MHC class II antigen processing. The MIIC is the MHC class II compartment, where proteases degrade endocytosed proteins. IFI30 specifically breaks down disulfide bonds of these proteins. Image courtesy of Nature Reviews: Immunology.
Methods/Approach
The human protein sequence for IFI30 (ENSP00000384886) was attained through the Ensembl database. This protein sequence was used as a query in a BLAST against the whale shark protein database with the Georgia Aquarium Galaxy server. Predicted protein hits were chosen based on their alignment lengths, e-values, and % identities along with query coverage, and their sequences were obtained through the Galaxy server. These predicted protein sequences were then used as queries in subsequent BLASTs against the human protein database through NCBI.
Orthologs were searched for by using the human protein sequence for IFI30 (ENSP00000384886) as the query sequence for BLASTs against the protein databases of other species.
A phylogenetic tree was created with CLUSTALW using the top predicted protein hits of the BLASTs against the protein databases of other species as well as the whale shark using the human protein sequence as the query. The best hits were recorded and analyzed.
Searching for IFI30 in the Whale Shark
The human IFI30 protein sequence was used as the query in a BLAST against the whale shark protein database. The BLAST resulted in only two predicted proteins as shown in Figure 2 and Table 1.
Figure 2
Figure 2: The results from a BLAST using the human IFI30 protein sequence as the query against the whale shark protein database through the Georgia Aquarium Galaxy server.
Table 1
The g13114.t1 whale shark sequence was used as the query in a BLAST against the human protein database. The best hit was a coiled-coil domain-containing protein 71 [Homo sapiens] (NP_075054.3) with a query cover of 85%, an e-value of 2e-19, and a percent identity of 52%. The g37936.t1 whale shark sequence was also used as the query in a BLAST against the human protein database, but the best hit did not contain any putative conserved domains. This whale shark sequence was also used in subsequent BLASTs against the elephant shark, zebra fish, and clawed frog protein databases but returned no putative conserved domains and no similar proteins matches for best hits.
The g13114.t1 whale shark sequence was also used as the query in a BLAST against the elephant shark protein database in order to further establish its relationship to the coiled-coil domain. The best hit matched the best hit from the BLAST against the human protein database. The elephant shark predicted protein hit, coiled-coil domain containing protein 71 (XP_007888756.1) had a query cover of 83%, an e-value of 2e-28, and a percent identity of 66%. This elephant shark sequence was then used as the query in a BLAST against the human protein database. This BLAST resulted in the same best hit in the human protein database as the BLAST with the whale shark sequence as the query.
Protein Domains
Since only the g13114.t1 whale shark protein sequence returned hits against the human and elephant shark protein databases with the coiled-coil domain conserved, it seems likely that whale sharks have the coiled-coil domain (as seen in Figure 3).
Figure 3
Figure 3: The putative conserved domain resulting from a BLAST using the g13114.t1 whale shark protein sequence against the human protein database.
Coiled Coil-type proteins are a structural domain of proteins. They usually consist of two or more alpha helices in a helical twist (Coiled coil, 2014). They are involved in many important biological functions, primarily the regulation of gene expression. They are also involved in HIV infection. A spring loaded mechanism constructed of gp41 protein forms a coiled coil, essentially forming a "trimer of hairpins" (or six helix bundle) that facilitates membrane fusion by pulling cell membranes closer to each other. The HIV virus then enters the cell and begins to replicate (Nash 2015).
Innate immune sensors that detect foreign molecules consist of N-terminal coiled coil, or TIR (toll/interleukin-1 receptor) domains along with LRRs (leucine-rich repeats) (Rairdan 2008). Coiled-coil proteins are involved in downstream signaling for the immune system (Nash 2015).
Orthologs
In order to find IFI30 orthologs in other species, the human IFI30 protein sequence was used as the query in BLASTs against the protein sequences of mice, zebra fish, elephant sharks, clawed frogs, yeast, and fruit flies. The best hits for each of the BLASTs are shown in Table 2.
Table 2
Phylogeny
A phylogenetic tree (Figure 4) was constructed through ClustalW using the best hits from the different species listed in Table 2 from the BLAST searches with the human IFI30 protein as query sequence.
Figure 4
Figure 4: A phylogenetic tree showing the best hits of the human IFI30 protein in mice, elephant sharks, clawed frogs, zebra fish, fruit flies, whale sharks (g13114.t1), and yeast.
It is interesting that the whale shark sequence g13114.t1 groups in between the fruit fly and yeast. The top hits from the fruit fly and the yeast protein databases were used as query sequences in BLASTs against the human protein database. The top hits yielded coiled-coil domain containing proteins. Therefore, we were confident that whale sharks conserved this protein domain. However, its distance from the human protein sequence as shown in Figure 4 implies that the whale shark predicted protein sequence is not very similar to the human IFI30 protein sequence.
Conclusions
We were not able to identify an ortholog of the IFI30 protein in the whale shark, but we were able to identity a conserved domain: the coiled-coil domain. Since the BLAST against the human protein database using the g13114.t1 whale shark sequence as a query did not return the IFI30 protein sequence as the best hit, we were not confident that the whale shark has this specific protein as an ortholog. However, we were able to identify the conservation of the coiled-coil domain through subsequent BLASTs with the elephant shark protein database, and we were able to establish a relationship. Therefore, whale sharks may not benefit from the activity of gamma-interferon-inducible lysosomal thiol reductase, which facilitates protein degradation. Further research could look into the way the whale shark's immune response is different from humans since it seems to lack the gene that encodes for the lysosomal thiol reductase.
References:
Balce, D. R., Allen, E. R., McKenna, N., & Yates, R. M. (2014). γ-Interferon-inducible Lysosomal Thiol Reductase (GILT) Maintains Phagosomal Proteolysis in Alternatively Activated Macrophages. The Journal Of Biological Chemsitry, 289(46), 31891-31904. doi:10.1074/jbc.M114.584391
Coiled coil. (2014, November 11). Retrieved April 13, 2015, from http://www.uniprot.org/help/coiled
Rairdan, G. J., Collier, S. M., & Sacco, M. A. (2008). The Coiled-Coil and Nucleotide Binding Domains of the Potato Rx Disease Resistance Protein Function in Pathogen Recognition and Signaling. Plant Cell, 20(3), 739-751. Retrieved April 13, 2015.
Maekawa, T., Cheng, W., & Spiridon, L. N. (2011). Coiled-coil domain-dependent homodimerization of intracellular barley immune receptors defines a minimal functional module for triggering cell death. Cell Host Microbe, 9(3), 187-199. Retrieved April 13, 2015.
Nash, P., Lin, D., & Binns, K. (2015). The Pawson Lab - CC Domain. Retrieved April 13, 2015, from http://pawsonlab.mshri.on.ca/index.php?option=com_content&task=view&Itemid=64&id=213
Neefjes, J., Jongsma, M., Paul, P., & Bakke, O. (2011, December). The basic MHC class II antigen presentation pathway [Jacques Neefjes, Marlieke L. M. Jongsma, Petra Paul & Oddmund Bakke]. Retrieved April 14, 2015, from http://www.nature.com/nri/journal/v11/n12/fig_tab/nri3084_F3.html
[Image]
West, L. C., & Cresswell, P. (2013). Expanding roles for GILT in immunity. Current Opinion in Immunology, 25(1), 103-108. doi:10.1016/j.coi.2012.11.006
Websites used for research:
http://www.ensembl.org/index.html
http://www.genome.jp/tools/clustalw/
http://blast.ncbi.nlm.nih.gov/Blast.cgi
http://whaleshark.georgiaaquarium.org/root/index